Two novel PCR-based assays for sexing of Silene latifolia and Silene dioica plants

Silene latifolia and S. dioica are model systems in studies of plant reproduction, chromosome evolution and sexual dimorphism, but sexing of plants based on morphology is only possible from flowering stage onwards. Both species show homogametic females (XX) and heterogametic males (XY).• Here we developed two assays (primer pairs ss816 and ss441) for molecular sexing of S. latifolia and S. dioica, targeting length polymorphisms between the X and Y-linked copies of the spermidine synthase gene.The two assays were successful in identifying known (flowering-stage) males and females from UK and Spanish populations, with an error rate of 3.1% (ss816; successful for both species) and 0% (ss441, only successful for S. latifolia). Our assays therefore represent novel tools for rapid, robust and simple determination of the genotypic sex of S. latifolia and S. dioica.


Background
Silene latifolia and S. dioica both display an active Y-system of sex determination, with heterogametic males (XY) and homogametic females (XX) [ 1 ].Hence, the two species have become model systems for plant sex determination and sex chromosome evolution [ 1-

Table 1
Details of primer pairs designed for sexing Silene .Primer name refers to its 5 ′ start position in the S. latifolia spermidine synthase DNA sequence from Genbank.Fragment length (bp) is the expected size in base pairs of the band for the Y and X copies.3 ].Silene species are also commonly used in studies of sexual dimorphism (e.g., [ 4 , 5 ]), although life history stages prior to flowering are rarely investigated [ 6 ].Therefore, the ability to sex an individual plant already prior to flowering would e.g.enable studies of sex allocation and sex-specific physiological processes.Unfortunately, existing molecular markers for sex determination in Silene are sub-optimal.Methods such as those in Zhang et al. [ 7 ] and Mulcahy et al. [ 8 ] often require post-PCR steps and lack additional fragments that amplify in both sexes to act as a control, potentially limiting their usefulness [ 9 ].More recently, Hobza and Widmer [ 10 ] described a PCR-based method of sexing S. latifolia and S. dioica .However, we found this method to be unsuccessful in our laboratory when tested on UK populations, conceivably a result of inter-population differences on the sex chromosomes [ 11 ].We therefore developed a novel molecular method for sex determination in S. latifolia and S. dioica , aiming to obtain a rapid, cheap and robust assay.

Plant material
UK populations of S. latifolia and S. dioica were commercially ordered as seeds from Chiltern seeds (Wallingford, England, UK) then sowed and grown Cardiff University Talybont Glasshouses (51.500210, -3.200921).Spanish S. latifolia seeds were collected from 5 natural populations in Galicia, Northwest Spain (see [ 5 ]).

DNA extraction
One ca.75 mm 2 disc of fresh leaf tissue was ground in 1.5 mL microcentrifuge tubes using micropestles.400 μl of Edward's extraction buffer (200 mM Tris, 250 mM, 25 mM EDTA, 0.5% SDS) was added to ground samples.The tube was vortexed and incubated at 55 0 C for 10 min.The extract was centrifuged at 13,000 rpm for 1 min and 300 μl of the supernatant transferred into a sterilised Eppendorf tube.The supernatant was mixed with 300 μl 100% isopropanol and left at room temperature for 2 min.After centrifugation at 13,000 rpm for 15 min, the supernatant was removed and discarded and 300 μl of 70% ethanol was added to the pellet in the tube and left for 3 min.Following centrifugation at 13,000 rpm for 5 min the supernatant was removed and discarded, and the pellet was left to air dry for a few minutes.Finally, the pellet was dissolved in 100 μl of TE buffer (10 mM Tris, 0.1 mM EDTA), incubated at 55 0 C for 30 min, and stored in a freezer (-20 0 C) until required.

Polymerase chain reaction (PCR)
Fragments were PCR amplified in 15 μl reaction volumes containing: 0.25U GoTaq G2 Flexi DNA polymerase (5 U/μl, Promega), 1x Promega Green GoTaq Flexi Buffer; 0.4 μM of each primer, 2.5 mM MgCl 2 , 167 μM of each dNTP along with 1 μl of DNA extract.PCR reactions started with 3 min at 95 0 C followed by cycling of 95 0 C for 30 s, 61 0 C ( ss816 ) or 50 0 C ( ss441 ) for 75 s, 72 0 C for 45 s, for 38 cycles, then a final 10-min extension step at 72 0 C. PCR products were run on 3% agarose gels at 120 V for 65 min, stained with SYBR TM Safe (ThermoFisher, UK) and viewed under UV light.Negative controls were included in each reaction to monitor for any contamination.

Development of Silene sexing primers
Primer design was based on a sex-linked gene identified by Filatov [ 3 ] with differentiated copies on the X and Y chromosome: S. latifolia spermidine synthase gene (GenBank accessions: AY705437.1,AY705438.1;[ 3 ]).Two primer pairs ( Table 1 ) were designed, targeting areas flanking insertion/deletion events of the X or Y chromosome sequences, resulting in the Y gene copy being visibly shorter on agarose gel than the X copy, for robust sex diagnosis [ 9 ].

Method validation
When applied to known-sex plants, both assays ( ss816 and ss441 ) generally provided the predicted outcomes for sexing: genotypic females displayed only the X-specific band, while genotypic males additionally showed the Y-specific band ( Figs. 1 A; 2 A).When using assay ss816 to sex 98 individuals, including samples from UK and Spanish populations of S. latifolia and UK populations of S. dioica , results matched the phenotypic sex in all but 3 cases ( Table 2 , and see below), corresponding to an error rate of 3.1%.While it does not affect the ability of accurate molecular sexing, it should be noted that in the Spanish population of S. latifolia the X-specific band only amplified in females, but not in males -still yielding the male-specific band in males, and the X band as an amplification in females

Table 2
Summary of PCR amplifications of each primer pair assay.Column 'Unknown sex' indicates individuals with unknown phenotypic sex that were screened using our sexing assays.The error rate was calculated by dividing the number of unsuccessful/mismatched individuals by the total number of known-sex individuals tested.( Fig. 1 B).In addition, using ss816 we obtained clear test results for 163 S. latifolia individuals, with the test providing non-ambiguous results as either male or female for each tested sample.However, since their phenotypic sex was unknown, this merely demonstrated that the assay gave clear, albeit unverified, results for a relatively large number of individuals.Use of ss441 to sex 55 individuals from the same populations yielded assay results that matched the phenotypic sex without any mismatches, giving an error rate of 0% ( Table 2 ).For this assay, S. dioica did not reveal discernible differences between males and females ( Fig. 2 C), leading to the inability to sex individuals.An additional 40 S. latifolia individuals with unknown phenotypic sex successfully amplified for ss441 .Of these 40, 35 matched the sex predicted by assay ss816 , and the remaining 5 were solely amplified by ss441 .The three individual mismatches in ss816 between the genotypic and phenotypic sex (two in S. latifolia and one in S. dioica ) were all phenotypic males (exhibiting male flowers), yielding the assay outcome of a female.These same individuals did not amplify in the ss441 assay.These results were confirmed by re-extracting DNA and repeating the PCRs.Contamination could not explain the outcome, since a Y band was missing, rather than an extra band occurring.The three observed mismatches do not necessarily discredit the accuracy of our assays, for three reasons.Firstly, the lack of clear amplification of ss441 could suggest issues with the quality/quantity of the DNA extracts.Secondly, the target gene spermidine synthase is sex-linked, but not an area involved in sex determination [ 3,12,13 ].Therefore, the expression of the phenotypic sex is independent of the presence of the region amplified by both primer pairs.Deletions in this gene could therefore explain our observations, consistent with the observed degeneration of Silene latifolia Y chromosomes [ 14 ].Thirdly, it could also be suggested that the primer target sequence on the Y chromosome shows point mutation polymorphisms among populations [ 11 , 14 ], resulting in unsuccessful primer binding and failed amplification for some populations.Future work on sex chromosome polymorphism in Silene studies may reveal further details on the apparent mismatches between the sex inferred from flowers versus Y-chromosomal assays such as ours.Similarly, the observed relatively high inter-population differentiation for Y-linked genes in S. latifolia [ 11 ] could also explain why Spanish and UK populations of S. latifolia varied in assay suitability.It could be that the primers targeted an area of high mutation rates on the Y and X chromosomes leading to varied results of amplification, differing between and within species.
In conclusion, assay ss816 successfully amplified the X and Y chromosome sequences of spermidine synthase in individuals of both Silene species, and ss441 was successful for S. latifolia .Our assays can be used alone or in tandem to identify genotypic males and females of S. latifolia in UK and Spanish populations, as well as the related S. dioica, aiding ecological and genetic research.We anticipate that apparent 'mismatches' such as these found in the present study may in fact reveal interesting aspects of sex chromosome evolution in Silene .